Cynthia A. Challener is a contributing editor to Pharmaceutical Technology.

Cynthia A. Challener

	During biosimilar development, it is necessary to demonstrate that the physicochemical properties, efficacy, and safety (e.g., immunogenicity) of the biosimilar are similar to those of the targeted branded biologic. Therefore, in addition to structural and biochemical analyses of biosimilars for confirmation of the physical and chemical characteristics of the biosimilar, it is necessary to demonstrate that the pharmacodynamics (PD), pharmacokinetics (PK), and immunogenicity of the biosimilar are also comparable to those of the branded drug. Consequently, functional assays, such as ligand-binding assays (LBAs) and mechanism of action (MOA) cell-based assays (CBAs), must be developed to effectively assess biosimilars. In many cases, LBAs are crucial for determining PK and PD behavior, and they are also often used as orthogonal assays to CBAs.


	Most biotherapeutics rely on specific binding activities to achieve a clinical effect. Therefore, matching the binding activities of a biosimilar candidate to those of the reference product is part of the process of developing a biosimilar and something that is expected by both the European Medicines Agency (EMA) and FDA, according to Gino Grampp, R&D policy director for Amgen. Physicochemical assays for testing of originator and biosimilar molecules, however, can only assess the actual structure of the molecules, and do not provide information on the functional conformation of the molecules and how they interact with the intended target. “Only a functional assay, such as an LBA or CBA, can fully address this issue,” says Michael Sadick, senior manager for large molecule analytical chemistry with Catalent Pharma Solutions. 

	For monoclonal antibodies (mAbs), which make up a large portion of the biosimilars currently under development, it is important to remember that the different ends of the molecule are involved in binding with different activities (the Fab and Fc regions, which bind to the antibody target/ligand and Fc receptors [CD64, CD32, CD16, and FcRN], respectively). While 

 biological activity assessments provide vital holistic information on biosimilarity, a combination of ligand- and Fc-binding assessments may provide more sensitive and mechanistic values for biosimilarity, according to Sadick.

	FDA, in its May 2014 draft guidance on how to show biosimilarity using clinical pharmacological data, indicated that LBAs, concentration and activity assays, and PD assays were important for biosimilar product development. In particular, according to Grampp, FDA suggested in the 2014 guidance that analytical assays should be able to specifically detect active drug, for example, via binding assays that use reagents specific for the binding sites for the drug (e.g., the complementarity determining regions [CDRs] of an antibody). “In this case, the binding assay is used not just to detect the molecule concentration, but to test the in-vivo properties of the molecule, and therefore, a well-designed and sensitive assay can be of great benefit to a biosimilar developer,” Grampp observes. 

	In October 2014, the American Association of Pharmaceutical Scientists (AAPS) Ligand-Binding Assay Bioanalytical Focus Group Biosimilars Action Program Committee published a whitepaper with recommendation for the development and validation of ligand-binding assays for PK assessments. Sadick notes that the AAPS whitepaper has similar conclusions regarding LBAs for bioanalytical testing as appear in the FDA 2014 guidance. “Biosimilarity testing for bioanalytical samples [PK/PD studies] is pretty much fully dependent on ligand-binding analyses. Binding assays are useful for not only assessment of the similarity of the final structures of the originator and biosimilar molecules, but also for assessment of the similarity of the antigenicity of the originator and biosimilar molecules. Binding assays work very well for the detection of antidrug antibodies, and for testing which of those antibodies may have drug-activity-neutralizing potential,” says Sadick.


	Binding activities can be determined by either cell-based assays or ligand-binding assays. Ligand-binding assays are of great importance for the analysis and comparison of the originator and biosimilar therapeutic for both testing of the drug before administration to preclinical or clinical recipients, as well as for testing of an active drug in preclinical or clinical recipient sera, according to Sadick. The latter would include both assessment of the function of the drug in the serum, as well as the activity of potentially induced host neutralizing anti-drug antibodies (nAbs). “Very importantly, for bioanalytical studies there are very few cell-based bioassays capable of working with sera derived from preclinical or clinical recipients. Thus, for bioanalytical assessments of biosimilarity, ligand-binding assays are in many cases the only functional assays available,” Sadick states. 

	In addition, ligand-binding assays enable the characterization of the potency of different biosimilars and provide crucial information on the affinity (dissociation rate constant, KD) and selectivity of biosimilars to specific targets, according to Roger Bosse, senior product leader for research reagents with PerkinElmer. He adds that regulatory authorities recommend simple and reliable methods for the characterization of biosimilars, and LBAs are one type of alternative.

	For drug substance/drug product testing, on the other hand, Sadick notes that cell-based functionality is suggested whenever possible. “For ligand-type therapeutic molecules (e.g., erythropoietin), ligand-binding assessments should probably not be used as a first choice for biosimilarity testing, because cell-based assessments are more biomimetic to the ligand mechanism of action as compared to ligand-binding assays and tend to be more sensitive to small differences or changes,” he says. If a cell-based bioassay cannot be established, however, Sadick indicates that ligand-binding assays would be the first choice for biosimilarity assessment. 

	In addition, for mAbs, ligand-binding assays do provide a valuable assessment of biosimilarity and should be included, orthogonally, with cell-based functionality assessments.


	It is, in fact, important to remember that the totality of evidence-a stepwise approach to evidence development that includes both structural and functional analyses-will ultimately be required to address residual uncertainties and establish biosimilarity, according to Margaret Karow, executive director of biosimilars process development at Amgen. “While target binding assays are a critical component of the analytical comparison of biosimilars to their corresponding reference products, the assays do not replace other analytical assessments required, nor do they provide information about the activities of the molecules, such as cellular signaling or effector functions (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Furthermore, the assays cannot predict the extent of clinical benefit or potential differences in safety, such as those identified risks for the reference product or unidentified risks for the biosimilar, and lastly, they are not predictive of a molecule’s potential immunogenicity,” she states.


	There are essentially two general methods for assessing ligand binding: ELISA (enzyme-linked immunosorbent assay)-type assays and kinetic binding assays. Classical ELISA assays were based on horseradish peroxidase or alkaline phosphatase and relied on color detection using a microplate colorimeter, which often suffered from poor sensitivity, particularly for samples containing serum, according to Sadick. Today, he notes that the final reagent/moiety of an ELISA test can be analyzed using several other techniques, including time-resolved fluorescence, electrochemiluminescence, and proximity-bead-based assays. “Each of these assay approaches provides differing strategies for increased sensitivity and decreased background or serum interference,” Sadick says. Matthew Reuter, biotherapeutics business development with PerkinElmer, adds, “These novel nonradiometric homogenous no-wash platforms aid in assay transferability, reproducibility, scalability, and overall cost reduction. They are also very robust and meet development criteria, which is equally important because typically LBAs are developed by upstream analytical method development groups and subsequently transferred to downstream QC-GMP lot release groups.”

	Ligand-binding kinetic assays for biosimilarity testing typically rely on surface plasmon resonance (SPR), according to Sadick. SPR measures binding in terms of the “change in mass” that occurs as the ligand and binding partner attach to one another and then separate. Advances in SPR analysis have resulted in increased robustness of the assays and reduced operator influence, as well as increased throughput for the determination of binding affinities. Biolayer interferometry (BLI) is a newer method that, like SPR, measures binding in terms of the “change in mass,” but uses eight biosensor tips rather than microfluidics. BLI also allows for hybrid binding for quantitative ELISA-like binding determinations, which may be applicable for PK/PD bioanalytical analyses, according to Sadick.


	One of the biggest challenges with LBAs is determining when they are appropriate. “LBAs are certainly easier for starting a development campaign, but recent regulatory guidelines indicate that a cell-based MOA bioassay should accompany the development of a drug candidate as early as possible,” says Reuter. If an LBA is used for determining the MOA or bioactivity of a molecule, it must eventually be translated to a cell-based format, and therefore it is necessary to demonstrate perfect correlation between the LBA and the cellular assay. “Adapting traditional complex cellular functional assays into simpler binding assays that rely on purified cellular components (e.g., cell surface receptors) is another important challenge,” Reuter adds. PerkinElmer is developing both binding and functional assays for characterization and cell-based determination of the MOAs of biological drugs (including biosimilars) that bind to specific cell-surface receptors, antigens, or soluble ligands using various technologies. 

	Target solubility is another issue, according to Karow. “High-throughput, kinetic assessment of target binding on cells for those targets that cannot be produced as soluble proteins remains a challenge for the industry,” she observes. Several companies are, however, developing technologies designed to overcome this problem, including high-throughput, high-performance, real-time, label-free analysis systems.

	For Catalent, the need to derivatize therapeutic mAbs, or the “test” moieties, to immobilize them has been a concern. “It is more optimal to immobilize the receptors so that the mAb samples could be analyzed without any derivatization,” says Sadick. Therefore, the company immobilized all six recombinant Fc receptors on nickel-coated biosensor tips for use in BLI analyses. “With this approach to binding kinetic analysis, the KD values for mAbs, both originator molecules and biosimilars, can be accurately and reproducibly determined,” he asserts.

Cynthia A. Challener, PhD, is a contributing editor to Pharmaceutical Technology.

When referring to this article, please cite it as:
	C. Challener, " Chiral Resolution with and without Resolving Agents," 

Articles

Ligand-binding assays are fundamental to characterizing biosimilars.

Ligand-Binding Assays and the Determination of Biosimilarity

	Approaches to drug development are changing, as pharmaceutical companies use advanced technologies to achieve targeted goals. Today, rational drug development strategies based on modeling, simulation, and biomarkers complement such traditional drug discovery methods as structure-activity relationship (SAR) studies. Biomarkers are also being used to evaluate drug performance during preclinical testing and clinical trials. Assay development and validation, however, can be more challenging for biomarkers than it is for APIs. This article will examine some of these issues.
	
	Many of the challenges of working with biomarkers stem from the fact that, unlike APIs in neat solutions, they are endogenous compounds in biological fluids. For example, isobaric analytes such as bile acids in matrices can lead to higher background noise and interference in biomarker assays. In addition, biomarkers are typically present in trace amounts, unlike APIs, according to Stephanie Pasas-Farmer, senior director of bioanalytical labs with Frontage Laboratories.

	Furthermore, analytical method development for APIs tends to be more straightforward because reference materials with known concentrations are available and can be used to spike into the matrix. With biomarker assays, internal standards are not often available, and, generally, an endogenous level of the biomarker is already in the matrix, which makes quantitation a challenge, according to April Brys, director of biomarker services with Battelle.

	Regulatory requirements for analytical methods also differ for APIs and biomarkers. API analytical method development and validation must meet US Pharmacopeial Convention requirements or guidelines set by the International Conference on Harmonization (ICH).

	Biomarker assay development and validation, meanwhile, depends on the assay’s intended use. If it is being used as a diagnostic tool, it must meet requirements of the Clinical Laboratory Improvement Amendments (CLIA).  If it is being used to establish a pharmacokinetic/pharmacodynamics (PK/PD) endpoint, it must meet good laboratory practice (GLP) standards.

	Many of the same analytical techniques, however, are used for API methods and biomarker assays. “Small-molecule biomarker assays are typically developed using a liquid chromatography/mass spectrometry (LC/MS) platform, which provides a great balance of sensitivity, selectivity, robustness, precision, accuracy, and ease of operation,” states Patrick Bennett, executive director of biomarker operations at PPD. The LC/MS platform is also beneficial for biomarker analysis because it can be used to perform multi-component analyses simply, including metabolomics-based analyses of hundreds of molecules per sample.
	
	


	For biomarker assays, regulatory agencies prefer that calibration standards and quality control samples be prepared using the same biological matrix (whether blood, urine, plasma, or other biological fluid) found in the samples to be analyzed. However, they should be free of biomarker, which can influence the accuracy of readings. Unfortunately, it is difficult to obtain analyte-free matrices.
	
	One option, according to Pasas-Farmer, is to use a surrogate matrix that is biomarker free. A second option is to remove the endogenous analyte/biomarker from the matrix through chemical treatment, charcoal stripping, selective extraction, or some other method. In both of these cases, however, the chosen matrix will be different from the study sample matrix. “With surrogate, stripped, and depleted matrices, there may be issues with solubility, stability, non-specific binding, or ionization matrix effects due to the differences in the modified matrix and the authentic matrix,” says Bennett. Consequently, it will be necessary to design validation experiments to confirm that the ability to quantify analytes in a modified matrix is similar to that for analytes in the authentic matrix. If an authentic matrix is combined with standard addition, Bennett adds, experiments and processes will be necessary to determine the baseline and final concentrations and generate bridging data from newer lots.

	These issues may be avoided by using a standard addition approach with the true biological matrix once the basal levels of the endogenous biomarker have been calculated for the specific lot of control matrix. Stable, labeled reference material can be used as a surrogate standard, and labeled internal standards can be effective. These approaches, however, can also be challenging because, often, reference standards are not available, or are difficult to synthesize, for biomarker assays. Even if they are available, whether they are labeled or unlabeled, they can be expensive.

	In some cases, depending on the detection method and the type of matrix, samples may have to be purified before a biomarker assay can be completed. This, in turn, can create various problems, including recovery and matrix interference issues, notes Brys.
	
	


	Before method validation, it is crucial to identify the intended use of the biomarker assay. “The investigator must understand the ultimate use of the data generated,” says Pasas-Farmer. If biomarker data are to be used for early screening and evaluation of the mechanism of action (MOA) of a drug candidate, then a qualified assay that is semi-quantitative or relatively quantitative can be used, she says, but if the biomarker data are used to support claims for the safety/efficacy of dosing in a pivotal preclinical or clinical study, and if they will be used in a regulatory filing in support of a new drug application (NDA), then a fully validated assay should be implemented.
	
	Finally, if the assay will be used as a diagnostic tool to determine the disease state of an individual, then CLIA validation requirements must be met, she explains.

	Discussions are still ongoing, however, about whether biomarker assays should be fully validated, or if qualification of an assay can still be used if the new chemical entity is in the early stages of development. Bennett notes, for example, that, even if no compliance is deemed necessary, extensive method performance qualification may still be warranted to ensure that the biomarker assay can provide adequate confidence in the data required.

	Validation of an API analytical method is typically focused on the validation of specific assay performance parameters such as the precision, robustness, linearity, and lower limit of quantification (LLOQ)/lower limit of detection (LLOD). In contrast, validation of biomarker assays involves determination of “fitness for purpose” and must demonstrate that the assay meets the predetermined purpose, according to Brys, who defines the stages of biomarker validation as follows:

		Defining the purpose and selection of the assay

		Identifying the critical reagents and standards

		Writing the procedure and validation plan

		Performing the actual study validation, which typically includes assessment of fitness for purpose, robustness of the assay in the clinical setting, standardization of patient sampling, and the collection, storage, and stability of the samples.

		Evaluating the assay during routine use

		Identification of batch-to-batch issues.

	If stable labeled material is used as a surrogate reference standard, Bennett says, additional experiments must be performed immediately before each analytical run, to demonstrate similar responses for equimolar amounts of labeled and unlabeled analyte. Because biomarkers are endogenous and the goal is to measure the change in the biomarker concentration in response to some other change (e.g., use in diseased vs. healthy patients or in comparing different therapies), he says, experiments must also be run to determine the ability to detect a statistically meaningful change.
	
	


	While it might seem obvious, Bennett stresses that, even before assay development starts, it is crucial to ensure that the correct biomarker(s) is being evaluated for the right endpoint(s). Brys adds that selected biomarkers should have a strong biological rationale and correlation with efficacy in preclinical studies and, thus, focusing on a few biomarkers is more effective. “A basic starting point for the development of any biomarker assay is an understanding of relevant precedents for the assay,” agrees Donna Eash, director of client services with Frontage.
	
	As important is ensuring that the extent of qualification or validation is aligned with the objective of the biomarker data that is obtained, according to Bennett. “If the data are used for dose selection, formulation comparisons, or research, then compliance may not be as important as ensuring that the method meets an appropriate acceptance criteria. However, if the data are to be used for patient enrollment, safety, or efficacy, meeting required regulatory compliance standards also should be included in the strategy,” he observes.
	Developers must also outline an effective development plan, considering that endogenous biomarkers are often identified in discovery stages. “A good strategy must be in place for initial discovery into preclinical, followed by preclinical into clinical, as each stage will have its own level of rigor, and follow the fit-for-purpose assay qualification and validation steps,” Eash notes.

	Given the low levels of biomarkers present in most samples, Brys adds, it is important to identify an analytical method with sufficient sensitivity. Access to actual study or patient samples can be important during the pre-qualification/validation stage to ensure that the right concentration range(s) are being evaluated and to determine differences in the concentrations of the biomarker(s) in treated and untreated samples, according to Bennett. He also reiterates that validation experiments should be performed to quantify any matrix effects and non-specific binding that may occur when using modified or surrogate matrices.

	The following are also important to the successful development and validation of biomarker assays, Brys says:

		Evaluating biomarker stability, particularly during storage

		Proper determination and evaluation of sample collection and storage methods

		For biomarker assays that are intended for use in diseased patients, the validation process must also include screening patients to evaluate how the biomarker is affected by the disease state and how the disease state will affect the analysis, according to Pasas-Farmer.


	New advances in analytical technology, such as multiplexing systems that allow for 10–100 biomarkers to be analyzed in a single sample, offer a number of benefits, including lower costs and fewer invasive procedures, says Pasas-Farmer.
	
	High resolution/accurate mass (HR/AM) instruments are now commercially available with triple quadrupole-like sensitivity and robustness. Bennett says that they offer significantly higher resolving power with a much greater signal-to-noise ratio, even when using single-ion monitoring (SIM). SIM is advantageous because it does not require the fragmentation and subsequent loss of signal that occurs in LC-MS/MS experiments. This means that, for many analytes, particularly those with poor or excessive fragmentation or high background noise levels, even when MS/MS is used, it allows for greater selectivity and sensitivity, he says.  Examples would include bile, which has poor fragmentation, and steroids, with excessive fragmentation.

	Easier and more robust nanospray technologies have also allowed researchers to achieve significantly better LLOQs than was possible with high-flow liquid chromatography and traditional electrospray ionization techniques, Bennett says. Pairing this technology with either HR/AM or triple quadrupole instruments can provide sensitivity benefits that are further magnified when multidimensional chromatography and an immunoaffinity cleanup step are used, he adds.


	Regulatory expectations for biomarker methods are changing, and Bennett expects them to continue to evolve over the next three to five years. FDA’s recent guidance suggests that the agency will take a more rigorous and less semi-quantitative approach to overseeing assay qualification and validation. In addition, Incurred Sample Reanalysis, which is used to measure the quality of data and robustness of an assay and has been required for small- and large-molecule drug substances, is now being requested for biomarker development, Pasas Farmer points out.


	Cynthia A. Challener, PhD, is a contributing editor at 

 When referring to this article, please cite it as C. Challener, “Developing and Validating Assays for Small-Molecule Biomarkers,” 

Working with biological matrices and understanding the intended use are crucial.

Developing and Validating Assays for Small-Molecule Biomarkers

	Single-use systems (SUS) have become established in biopharmaceutical drug development. As confidence in the technology has increased, the adoption of disposable equipment for commercial manufacturing has begun to take place. With this shift has come a dependence on suppliers that is related to production capabilities, a situation that did not previously exist. This reliance on suppliers translates to increased risk, and consequently biopharmaceutical manufacturers are focused on managing this risk and ensuring security of supply. Dual sourcing of single-use systems and components was initially proposed as the key solution. After further consideration of the issues surrounding the supply of single-use systems, however, many in the industry believe that dual sourcing presents many challenges that can often be avoided by taking alternative approaches.
	
	


	Today, single-use systems are used throughout the entire process chain from upstream to downstream and from bench scale to production scale. In the past, when drugs were manufactured in stainless steel, the manufacturer was in control of the infrastructure once its facility was in place, sourcing only critical disposable components such as filters and chromatography sorbents. Although disposable systems do provide numerous advantages, such as reduced contamination risks, shorter set-up times, and lower capital costs, biopharmaceutical manufacturers do not have that same level of control over the disposable systems as they did with traditional stainless-steel equipment because the systems and components must be purchased from a supplier, according to John Briggs, director of quality, regulatory, and compliance at ASI-Life Sciences. As a result, adds Mario Philips, vice-president of single-use technologies at Pall Life Sciences, there is a greater dependence on the single-use supplier, which increases the risk for the manufacturer and thus requires greater vendor transparency and supply-chain security.
	
	“Unlike with traditional equipment, where almost everything is physically located at the facility, single-use systems are delivered to the site from a supplier. If the user operates on a just-in-time basis with no inventory, any delay in delivery could put the manufacturing schedule at risk. Consequently, an SUS is a critical part of drug substance manufacturing, and there is a greater need to understand and secure the sourcing of these systems,” says Roman Rodriguez, global market development manager for single-use technologies with EMD Millipore. As such, it is logical to consider qualifying a back-up supplier as part of the SUS risk-management system, and dual sourcing is a response to a need for anticipating unforeseen issues and scenarios.
	
	The implementation of single-use systems in commercial manufacturing has had the additional consequence of shifting the purchasing of these systems from those in development to sourcing professionals. As a result, according to Tony White, a founding director of the BioPhorum Operations Group (BPOG), some biopharmaceutical manufacturers are now starting to consider single-use technologies as a strategic commodity and managing their purchases across their manufacturing network, which often leads to the identification of risks that were not previously perceived. “In addition,” says White, “they are realizing that they have been single-sourcing key components by default--a position they do not find very comfortable.”
	
	Past experience with SUS sourcing problems is also a driver for the increasing focus on multisourcing, according to Terry Hudson, director of process development engineering with Genentech. “At any conference with single-use as a topic, you can find a case study or conversation about a single-use component being changed with either no prior notification or inadequate notification to the end user, and in several cases that has been tied to a negative impact on production,” he explains. In addition, Hudson notes that companies are also learning that occasionally a given process may be more sensitive to a leachable or film type than other processes, and it is beneficial to have an alternative film that can be used with that process.  
	
	There are regulatory drivers as well. Regulatory authorities have had a clear and simple message with respect to single-use products, according to Ross Acucena, product strategy manager-ReadyToProcess with GE Healthcare Life Sciences, which is that their use is analogous to outsourcing crucial elements of GMP operations, such as presterilization. “The role of the supplier is therefore now more critical, and regulators expect drug manufacturers to mitigate any risk with close partnerships and thorough knowledge of their suppliers’ quality systems and supply chains,” he says. Furthermore, the increased focus on suppliers has highlighted the fact that SUS supply chains are both broad and deep because of the integration of many components into the end products. “Single-use customers recognize that it is difficult to fully control the supply chain, which presents a potential risk to their operations and subsequent supply of needed drugs to patients,” Acucena asserts. The industry response has been to increase its focus on mitigating such risk through multisourcing.  
	
	


	The ultimate goal of multisourcing is to have the ability to seamlessly transfer from one single-use product source to another without risk to patient safety, according to Acucena. “There is a strong need to characterize and understand this issue for all parties in the complex supply chains that exist within the industry. Upon investigation, it may materialize that some parties have a robust supply chain where risk is small. The converse is that unacceptably high levels of risk are detected, requiring either dual-source or multisource strategies, or even exiting a specific relationship,” he observes.
	
	In an ideal state, according to Hudson, the multisourced component would be supplied by two different companies with different supply chains to minimize the potential for a single change impacting both components. At a minimum, multisourcing needs to provide assurance that a change or disruption to one source will not also occur at the second source. Thus, risk mitigation can be achieved by using one vendor that has two plants or by using two separate vendors. “The latter option also provides some commercial leverage and tends to be the preferred approach for end users,” Philips says, “but a lack of industry standards for single-use systems makes it more difficult, if not impossible.” Not surprisingly, suppliers prefer the dual site or manufacturing redundancy concept, according to Spencer Parkinson, senior product manager for bioproduction with Thermo Fisher Scientific. “If it is truly assurance of supply that is of interest, then more than one production site addresses this need, while allowing a supplier to provide ‘best-in-class’ products,” he notes.
	
	For Rodriguez, multisourcing is the basic view, and the real issue relates to supply chain understanding, assurance, and control, as well as taking action to reduce inherent risks. “Doing so is not as easy as it seems due to supply chain complexity down to the resin suppliers and the varying approaches that can be taken. It really starts with a close interaction with SUS integrators on a whole supply chain view. An alternative supplier’s products must be assessed with the same science-based qualification process, and if different suppliers adopt slightly different approaches, the end-user must determine what is acceptable,” he says. As an example, Rodriguez points to bioburden levels, which can be tested on every single lot or using a statistical approach. Both methods may be acceptable depending on the end-user’s specifications.
	
	


	There are numerous challenges when considering true dual sourcing, and many relate to the complexity of the single-use supply chain. The SUS business is highly customized, and there are thousands of single-use components, many of which only work with other components made by the same supplier. Of course, not all those components need the same scrutiny depending on their criticality and demand, according to Rodriguez, but due to the huge number of components and suppliers, it is a complex task to track and understand the supply chain to secure and control it. As an integrator, EMD Millipore has prioritized and ranked SUSs based on criteria (i.e., criticality, volume used, lead time, reliability of the suppliers) and customer interaction.
	
	Qualification and validation are, in fact, the critical criteria that must be met to achieve dual sourcing, according to Acucena. “To achieve dual sourcing, one must have documented evidence that a change in input (product contact materials) will not affect the output (product quality, efficacy, safety), and it is ultimately through qualification and validation that such assurance is achieved,” he notes.
	
	ASI-Life Sciences is taking a close look at how to demonstrate functional equivalency of materials obtained from different suppliers. “At this point there is no standard or definition for functional equivalency, but it is necessary to define what constitutes functionally equivalent materials in order to ensure change control,” Briggs observes.

	“The challenge lies in the fact that proprietary plastics/elastomers are used by different suppliers, and thus composition alone cannot be used to determine equivalency, and other characteristics, such as the physical and chemical properties of the single-use product and its performance under process conditions must be evaluated. In addition, the ability to integrate the product into an end-user process and the quality system of the supplier must also be considered,” he comments.
	
	Standardization is frequently raised as another issue for dual sourcing of single-use systems. Philips notes that with limited standards in place, variation in SUS design continues to be significant, and it is much harder to have contingency in supply. Hudson agrees that the main challenge to dual sourcing is the current business model used by most single-use companies, which involves the development of hardware for a SUS that will only work with their single-use components. “This approach for end users is a significant impediment to deploying such systems into a GMP environment. Until companies are willing to standardize enough on their design that they fit more than one single-use assembly, including an assembly from another company, it will be difficult to achieve true dual sourcing,” says Hudson.  

	Parkinson, on the other hand, argues that standardization would lead to a halt in innovation. “Single-use products are not commodities and will not be in the foreseeable future; innovation is critical to improving the technology. While it also leads to the development of different designs and materials by each vendor, improvements would not occur otherwise” he states.
	
	The continued advancement of single-use technology adds to the complexity, however, with new products constantly introduced that require change management. “We are aiming at full process integration by adding supply chain considerations from the lead times associated with single-use items, which requires discussions with the end-user because it is an integral part of technology transfers into manufacturing operations,” Rodriguez says. “Obviously the primary action is to communicate and build a basis of understanding about the supply chain itself and the potential associated risks, which naturally leads to actions to minimize those risks at every level of the supply chain, starting with the single-use integrator. Focusing on the product attributes, quality, and specifications may not be enough, however, as there are other steps involved such as sterilization and transportation (sometimes international) that may impact the product and lead time. Ultimately, therefore, the first challenge is to get a clear understanding of the supply chain, to communicate about it, and then establish an action plan on a case-by-case basis,” he continues.
	
	Thoroughly understanding the supply chain is, however, an intense endeavor that requires significant resources, according to Acucena. He in particular points to the case where two SUS suppliers purchase equipment from the same precursor company, and thus qualifying the two SUS suppliers does not achieve dual sourcing. He also notes that there are significant costs associated with qualifying dual-source suppliers, and the value of such an investment is an important consideration. “In essence,” says Acucena, “dual-sourcing or multisourcing can be likened to an ‘insurance policy’ that is relatively expensive to buy.”
	
	


	While the initial reaction of many biopharmaceutical manufacturers to concerns over single-use supply chain security was to look at dual-sourcing solutions, the focus has since shifted to the broader issue of security of supply and how best to manage it, according to Acucena. “The need is security of supply,” agrees Kevin Ott, executive director of the Bio-Process Systems Alliance (BPSA). He adds that dual sourcing is one possible solution, but there are others with fewer validation challenges, including redundant supply sources from a single vendor and inventory plans, for example. “The question,” he continues, “also becomes complicated by how far back in the supply chain one needs to go. That depends on volume versus demand, structure of the supply chain, etc.”
	
	For example, Ott notes that suppliers of biocontainers that can assemble containers in two locations and carry sufficient inventory of finished units and film to cover film supply disruptions do not necessarily need to be dual sourced to ensure continuity of supply, just perhaps be “dual located.”
	
	“The justification for dual sourcing should depend on the risk and the level of simplicity, and single sourcing is in many cases appropriate as long as the relationship between the supplier and the end user is a partnership,” asserts White. He notes that many automotive manufacturers only single source strategically important materials because the investment in establishing a reliable supplier is significant and not worth doubling.
	
	One alternative to dual sourcing is, according to Philips, to limit the number of contact materials in single-use systems and aim to create interchangeability for any layer (e.g., films, filters, valves, and tubes) that comes in contact with a drug product. Pall Life Sciences has taken a step forward in this direction by vertically integrating its own production processes one step back in the supply chain. The company uses its own in-house extruded film to make the supply chain more robust and is working to mitigate any potential changes from resin suppliers. “While we can’t prevent change, we are prepared to manage that change, and if we fully understand our supply chain, we have the power to support customers through any change that may occur through our supply strategy, which adds up to an increased ability to offer a greater level of supply-chain security and continuity to users,” remarks Philips.
	
	Business continuity planning and inventory management are additional mechanisms for reducing risk, according to Acucena. He notes that suppliers are now engaged in an increased focus on these aspects, and thus the crucial first step of increasing industry awareness is already taking place. He also notes that the experience with reducing the risk associated with the single-sourcing of critical chromatography media, such as protein A, can be translated to single-use systems. “GE Healthcare sought to satisfy the industry’s need to mitigate this risk through a strong focus on business continuity planning (in accordance with ISO 22301 and achieving conformance to this standard), including qualifying dual sources for critical raw materials and a significant investment in safety stock. These efforts have delivered supply-chain security improvements that should help to eliminate end-user dual sourcing. Similar approaches should be applicable to single-use technology,” he explains.
	
	Both White and Ott also note that focusing on dual sourcing at this stage in the industry’s development may not be appropriate. “Many biopharmaceutical manufacturers are not yet in a good position to dual source, as they are just starting to understand the extent of their complete single-use portfolios across their networks,” White says. Ott adds that it may be premature or counterproductive to imagine risks. “It is better to actually study, evaluate, and understand your own supply chain and the real risks. Once that is done, the need to establish truly independent dual sources and the difficulty that such an approach represents with respect to validation and regulatory filing begins to look like much less of an issue than might be imagined,” he comments.
	
	


	Supply-chain security is a top challenge for both BPSA and BPOG moving forward into 2015. The Parenteral Drug Association (PDA) is also working on concepts to facilitate multisourcing. While these initiatives are important, Philips would prefer to see vendors, users, and regulators working together to bring one set of standards forward, rather than multiple associations working on multiple standards. “The biopharmaceutical industry is still a relatively immature industry, and substantial changes can be expected over the coming decade. Standardization is going to be a key element of true multisourcing, and having one body regulating the industry will make supply chains more transparent and keep user expectations realistic,” he says.
	
	Many of the industry-wide initiatives already underway to provide standardization and guidance on change notification, leachable/extractable testing, and other end-user needs for increased adoption of single-use systems, and particularly for components and systems that already allow some interchangeability such as buffer bags and tubing connectors, will help speed up multisourcing, according to Hudson. On the other hand, where the supplier’s business model is to design a system that only works with its components, education is the key. “Genentech recently presented a proof-of-concept design for a single-use bioreactor that could work with top- and bottom-mounted bags from two different suppliers. The next step is convincing management at suppliers that designs such as this one will lead to higher future sales because they help open the doors to more GMP applications and greater adoption of single-use technology in general,” he states. Genentech is also partnering with several single-use supplier companies to gain greater visibility into their supply chains, to provide recommendations on what it considers “multisourced”, and to communicate what applications of single-use simply will not occur until they can be multisourced.
	
	EMD Millipore, meanwhile, in addition to implementing a business continuity plan and supply risk management and emergency management programs focusing on products, suppliers, and its sites, respectively, has teamed with a third party (Resilinc Corporation) to map its supply chain for key products and suppliers to identify and mitigate risks with suppliers, according to Rodriguez.
	
	In its quest to identify a practical approach for determining the functional equivalency of unassembled components, ASI-Life Sciences submitted platinum-cured silicone tubing with similar specifications, mechanical properties, and performance profiles under typical use conditions obtained from two suppliers to Chemic Laboratories for comparative analysis of their extractable/leachable profiles. “Tubing was selected for this exercise because it is a common component of single-use systems and can be evaluated in a fairly straightforward manner,” Briggs observes. Importantly, no significant differences in the extraction profiles were observed for the two tubing samples. “These results indicated that the materials were comparable within the variability of the analytical techniques used,” Briggs says. He also recognizes that tubing is a universal single-use component, and establishing the functional equivalency of other, more proprietary single-use systems and components will be more challenging. “Our goal is to initiate a dialogue around the practical considerations for achieving dual sourcing of single-use systems.” 


	Vol. 38, Issue 12
	Pages: 24–26, 57
	Citation: When referring to this article, please cite it as C. Challener, "Securing the Single-Use Supply Chain," 

Dual Sourcing and the Single-Use Supply Chain

	Growing demand in emerging regions with simultaneous maturation of western markets has led to a new emphasis on low-cost, low margin products and specialized, low-volume niche drugs for targeted patient populations. To reduce costs and increase flexibility, many large pharmaceutical companies are outsourcing a growing percentage of their business activities. The changing market conditions are also creating opportunities for virtual pharmaceutical companies, which have no manufacturing capabilities of their own. In addition, increasing numbers of new drug candidates are highly potent, requiring specialized production capabilities. Custom manufacturing firms that have established themselves as efficient providers of services across the drug development and commercialization spectrum, and particularly those with specialized capabilities for the production of highly potent APIs, will be in a position to benefit as the trend toward pharmaceutical outsourcing continues.
	
	
	


	Market research firm Visiongain forecasts that the worldwide pharmaceutical custom manufacturing market will earn revenues of $71 billion in 2018 (1). Strong revenue growth is anticipated for market leading custom manufacturing organizations (CMO) between 2014 and 2024, with leaders including DPx Holdings, formed in 2014 from the contract manufacturing businesses of Royal DSM and Patheon; Lonza; and Catalent experiencing the greatest increase in absolute revenue. This growth reverses the trend seen in 2009-2013 of low single-digit revenue growth for the top five developed-market CMOs.
	
	During that same period, leading companies based in developing countries showed strong performance, with revenues increasing at double-digit growth rates, according to Visiongain  Not surprisingly, therefore, while North American and European CMOs have continued to dominate the contract manufacturing market in 2014, going forward, companies based in emerging countries will play a greater role as they experience sustained demand from domestic pharmaceutical companies and, increasingly, foreign drug developers. Specifically, between 2013 and 2024 emerging-market leaders will increase their share of the global CMO market by 15% (1).
	
	


	The increasing demand for CMO services in emerging markets reflects a wider trend in the pharmaceutical industry. The greatest growth in demand for pharmaceuticals is also taking place in emerging markets, but unlike in western countries where high-cost, high-margin sales were the norm, the buying habits are quite different, according to Diane Palmquist, vice-president of product management for GT Nexus, a provider of cloud-based solutions. “People in emerging regions purchase drugs by the dose, not by the bottle, and they are looking for low-cost (and consequently low-margin) products,” she says. “In order to remain successful, pharmaceutical companies have needed to completely transform their old business models,” she adds.
	
	In fact, during the past 10 years, demand for outsourced manufacturing services grew rapidly as pharmaceutical companies sought to take advantage of the benefits of contract manufacturing, such as lower costs, increased flexibility, and external expertise, while increasing their ability to focus resources on core competencies in drug development and marketing, according to Jonathan Weymer, senior pharmaceutical and healthcare industry analyst with Visiongain.
	
	Not surprisingly, in response to the changing market dynamics and in support of the desire to focus on core competencies, many large pharmaceutical companies have been consolidating their manufacturing facilities to reduce their asset bases and associated costs, according to Cristin Grove, director of contract manufacturing at Pfizer CentreSource. “In addition,” she notes, “as many products face loss of patent protection, they are transferred to contract manufacturers in order to allow the remaining captive capacity to be used for pipeline products.”  Pfizer CentreSource also anticipates real growth in the virtual pharmaceutical space, which will have a direct impact on the contract manufacturing sector, because these virtual companies typically have no manufacturing assets and require partners to help commercialize their opportunities.
	
	As a consequence, pharmaceutical companies are outsourcing nearly every aspect of the business, including API manufacturing. Palmquist notes that currently approximately 40% of industry business is outsourced, and she expects that number to increase to 80% over the next several years.
	
	


	CMOs are increasingly seen as strategic partners for pharmaceutical companies, providing a one-stop-shop of services for formulation development and manufacturing throughout the lifecycle of a drug, according to Weymer. “Market-leading CMOs have grown through acquisitions and site expansions and have positioned themselves to offer almost all required services on a global scale,” he says. It is important to note, however, that there is still a role to be played by specialist CMOs, particularly those that offer biological drug manufacturing services. Visiongain also found in its survey of the pharmaceutical industry that CMOs that can ensure supply chain quality control while also providing production flexibility are in greatest demand. “The ability to deliver high-quality, flexible production will drive business to the leading firms in the market in coming years,” Weymer observes.
	
	


	In addition to those CMOs that can offer integrated services from discovery through commercialization and off-patent production, contract manufacturers that have capabilities for the production of highly potent compounds will experience a higher share of the anticipated growth, according to Grove. “We believe there is significant upside in the high-containment space.  The primary driver is that pharmaceutical pipelines are focused on specialized disease areas such as the central nervous system (CNS), oncology, janus kinase (JAK) inhibitors, etc., and the vast majority of these new chemical entities (NCEs) require high containment.”
	
	While Pfizer CentreSource has access to Pfizer Global Supply sites with capabilities for containment of highly potent APIs, other leading CMOs such as Catalent and Lonza have made considerable investments in high-potency API and antibody drug conjugate production facilities in anticipation of long-term growth in demand (at least over the next 10 years), according to Weymer. Even so, with high containment facilities requiring substantial capital investments in excess of $50 million in many cases, this capital constraint will prohibit many pharmaceutical manufacturers and CMOs from adding this type of capacity, according to Grove.
	
	The key to success for highly potent API manufacturing will be for pharmaceutical manufacturers to partner early on in a project--such as Phase II/III--with CMOs that have the capability and capacity to produce highly potent compounds in development and commercial quantities. “With a partnership established in the earlier phases, it is much easier to continue and offer commercial manufacturing as well,” Grove explains. She sees Big Pharma and select (large) pure-play CMOs as the primary providers of this high potent API (and drug substance) capacity moving forward.
	
	Pfizer CentreSource’s current focus is on high containment solid oral dose and biologic drug substance offerings.  “We believe our globally integrated network, specialized manufacturing technologies, and quality/regulatory expertise will allow us to capitalize on the trend toward specialized disease areas,” Grove notes.
	
	

Pharma Leader Series: Leading Pharmaceutical Contract Manufacturing Organizations (CMOs) 2014-2024

 When referring to this article, please cite it as C. Challener, "API Custom Manufacturing Outlook: More Growth in 2015," 

Changing dynamics of the pharmaceutical industry are driving demand.

API Custom Manufacturing Outlook: More Growth in 2015

Dual sourcing is one of many possible solutions to securing the supply chain.

Securing the Single-Use Supply Chain

	With respect to FDA approval of new medicines, 2014 has been another strong year for the pharmaceutical industry. The agency’s Center for Drug Evaluation and Research (CDER) approved a total of 34 new medicines (as of the end of October 2014): 22 new molecular entities (NMEs) and 12 biologics license applications (BLAs) (see 

) (1). In 2013, the total number of approvals for the entire year was 27, while in 2012 the number reached 39, which was unusually high compared to the numbers of approvals over the previous 10 years (2). Not only were a high number of new medicines approved in 2014, there were numerous examples of first-ever approvals for the treatment of certain diseases and new classes of drugs that treat diseases via new routes. Treatments were also approved under new designations.


	Hetlioz (tasimelteon, Vanda Pharmaceuticals), a melatonin receptor agonist, was the first treatment approved by FDA for non-24-hour sleep-wake disorder, a chronic circadian rhythm (body clock) disorder that causes problems with the timing of sleep that occurs almost exclusively in people who suffer complete blindness (3). Vimizim (elosulfase alfa) from BioMarin Pharmaceutical is the first FDA-approved treatment for mucopolysaccharidosis type IVA (Morquio A syndrome), a rare, autosomal recessive lysosomal storage disease caused by a deficiency in 

 -acetylgalactosamine-6-sulfate sulfatase (GALNS). Vimizim is intended to replace the missing GALNS enzyme involved in an important metabolic pathway. Absence of this enzyme leads to problems with bone development, growth, and mobility (4). Myalept (metreleptin for injection), developed by Amylin Pharmaceuticals, which was acquired by AstraZeneca, is an orphan drug and the first treatment approved by FDA  for patients with congenital or acquired generalised lipodystrophy (5). Sylvant (siltuximab, Janssen Biotech) is another drug approved as a first treatment for a rare disease--multicentric Castleman’s disease, a rare disorder that causes an abnormal overgrowth of immune cells in lymph nodes and related tissues in the body. Administered as an injection, Sylvant works by blocking a protein that stimulates abnormal growth of immune cells (6).
	
	


	Several of the drugs approved by FDA also fall into new classes of drugs. Three of the most noteworthy are manufactured by Merck & Co. Zontivity (vorapaxar) tablets reduce the risk of heart attack, stroke, cardiovascular death, and need for procedures to restore the blood flow to the heart in patients with a previous heart attack or blockages in the arteries to the legs. It is the first protease-activated receptor-1 antagonist and is designed to decrease the tendency of platelets to clump together to form a blood clot (7). Belsomra (suvorexant) for insomnia is the first FDA-approved orexin receptor antagonist, which alters the signaling of orexin in the brain. Orexins are involved in regulating the sleep-wake cycle (8). Keytruda (pembrolizumab) was approved for the treatment of patients with advanced or unresectable melanoma who are no longer responding to other drugs. It is the first approved drug that blocks the programmed death-1 (PD-1) cellular pathway, which restricts the body’s immune system from attacking melanoma cells. Keytruda was also designated a breakthrough therapy and an orphan product and received priority review (9).


	Harvoni (ledipasvir and sofosbuvir) from Gilead Sciences is the first combination treatment (pill) for chronic hepatitis C virus (HCV) genotype 1 infection. It is also the first approved regimen that does not require administration with interferon or ribavirin, two FDA-approved drugs also used to treat HCV infection. Both drugs in Harvoni interfere with the enzymes needed by HCV to multiply. Harvoni was designated a breakthrough therapy (10).
	
	


	In addition, 2014 saw the first approved antibiotic drugs--Dalvance (dalbavancin, Durata Therapeutics), Sivextro (tedizolid, Cubist Pharmaceuticals), and Orbactiv (oritavancin, The Medicines Company)--designated as qualified infectious disease products (QIDPs) under the Generating Antibiotic Incentives Now title of the FDA Safety and Innovation Act (FDASIA) (11). Vimizin was also the first drug to receive the Rare Pediatric Disease Priority Review Voucher, a provision that aims to encourage the development of new drugs and biologics for the prevention and treatment of rare pediatric diseases (4).
	
	For only the second year, FDA was able to classify new NMEs and BLAs as breakthrough therapies, which are drugs with preliminary clinical evidence demonstrating that they may have substantial improvement on at least one clinically significant endpoint over available therapies. While in 2013 just three approved drugs were designated as breakthrough therapies, to date in 2014, six have already been approved: Zykadia (ceritinib, Novartis), Zydelig (idelalisib, Gilead Sciences), Keytruda, Harvoni, Ofev (nintedanib, Boehringer Ingelheim Pharmaceuticals), and Esbriet (pirfenidone, InterMune). Zykadia was approved for patients with a certain type of late-stage (metastatic) non-small cell lung cancer (12), and Zydelig was approved for patients with relapsed chronic lymphocytic leukemia, elapsed follicular B-cell non-Hodgkin lymphoma, and relapsed small lymphocytic lymphoma (13). Both Ofev and Esbriet were approved for the treatment of idiopathic pulmonary fibrosis, which is a serious, chronic condition in which the lungs become progressively scarred over time (14). Four additional supplemental submissions also received breakthrough therapy designations (15).
	
	In addition to the breakthrough designation, FDA also has several other designations designed to expedite the review of NMEs and BLAs. These include fast track, orphan product (for rare diseases), and priority review designations. Drugs can receive multiple designations. Up to Oct. 29, 2014, seven approved drugs had fast-track status, nine were designated as orphan products, and 12 underwent priority review (1).


	Looking at companies that received the most approvals, two have come out on top as of Oct. 29, 2014. Boehringer Ingelheim and Merck & Co. each received approvals for two NMEs and one BLA. Boehringer Ingelheim received approvals for Ofev, Jardiance (empagliflozin) for improvement of glycemic control in adults with type 2 diabetes, and Striverdi Respimat (olodaterol), a biologic drug for the treatment of chronic obstructive pulmonary disease (1). Merck’s winners included Zontivity, Belsomra, and Keytruda as described above. AstraZeneca, Eli Lilly, and Gilead Sciences were also successful with two approvals each: Gilead for one BLA and one NME (Harvoni and Zydelig, respectively); Eli Lilly for two BLAs, Cyramza (ramucirumab) for the treatment of stomach cancer and Trulicity (dulaglutide) for the treatment of adults with type 2 diabetes; and AstraZeneca for two NMEs, Farxiga (dapaglifozin) to improve glycemic control, along with diet and exercise, in adults with type 2 diabetes and Movantik (naloxegol) to treat opioid-induced constipation in adults with chronic non-cancer pain (1). Through its acquisition of the diabetes alliance assets of Bristol Myers Squibb, including Amylin Pharmaceuticals, AstraZeneca also acquired Myalept.
	
	


	1. FDA, “New Drugs at FDA: CDER’s New Molecular Entities and New Therapeutic Biological Products of 2014,” 

www.fda.gov/Drugs/DevelopmentApprovalProcess/DrugInnovation/ucm20025676.htm

, accessed Oct. 29, 2014.
	2. FDA, “Novel New Drugs 2013 Summary,” 

www.fda.gov/downloads/Drugs/DevelopmentApprovalProcess/DrugInnovation/UCM381803.pdf

, accessed Oct. 29,  2014.
	3. FDA, “FDA Approves Hetlioz: first treatment for non-24 hour sleep-wake disorder,” Press Release, Jan. 31, 2014.
	4. FDA, “FDA approves Vimizim to treat rare congenital enzyme disorder,” Press Release, Feb. 14, 2014.
	5. AstraZeneca, “US FDA approves orphan drug Myalept (metrleptin for injection),” Press Release, Feb. 25, 2014.
	6. FDA, “FDA approves Sylvant for rare Castleman’s disease,” Press Release, April 23, 2014.
	7. FDA, “FDA approves Zontivity to reduce the risk of heart attacks and stroke in high-risk patients,” Press Release, May 8, 2014
	8. FDA, “FDA approves new type of sleep drug, Belsomra,” Press Release, Aug. 13, 2014.
	9. FDA, “FDA approves Keytruda for advanced melanoma,” Press Release, Sept. 4, 2014.
	10. FDA, “FDA approves first combination pill to treat hepatitis C,” Press Release, Oct. 10, 2014.
	11. FDA, “FDA approves Orbactiv to treat skin infections,” Press Release, Aug. 5, 2014.
	12. FDA, “FDA approves Zykadia for late-stage lung cancer,” Press Release, April 29, 2014.
	13. FDA, “FDA approves Zydelig for three types of blood cancers,” Press Release, July 23, 2014.
	14. FDA, “FDA approves Esbriet to treat idiopathic pulmonary fibrosis,” Press release, Oct. 15, 2014.
	15. FDA, “CY 2014 Breakthrough Therapy Calendar Year Approvals,” 

www.fda.gov/downloads/RegulatoryInformation/Legislation/FederalFoodDrugandCosmeticActFDCAct/SignificantAmendmentstotheFDCAct/FDASIA/UCM380724.pdf

FDA approves treatments for new diseases and drugs that operate by new mechanisms.